Therapeutically useful polypeptides

ABSTRACT

DISCLOSURE RELATES TO THEREAPEUTICALLY USEFUL POLYPEPTIDES CONTAINING UP TO ABOUT NINETEEN AMINO ACIDS, PARTICULARLY TETRAPEPTIDES IN WHICH TWO BASIC AMINO ACIDS ARE JOINED TOGETHER THROUGH PROLINE. SUCH COMPOUNDS EXHIBIT VARYING DEGREES OF PHAGOCYTOSIS OR PINOCYTOSIS STIMULATION OR INHIBITION.

United States Patent 3,778,426 THERAPEUTICALLY USEFUL POLYPEPTIDES Victor A. Najjar, Cohasset, Mass., assignor to Research Corporation, New York, N.Y.

No Drawing. Filed Dec. 16, 1970, Ser. No. 98,890 Int. Cl. C07c 103/52; C07g 7/00; C08h 1/00 US. Cl. 260-1125 14 Claims ABSTRACT OF THE DISCLOSURE Disclosure relates to therapeutically useful polypeptides containing up to about nineteen amino acids, particularly tetrapeptides in which two basic amino acids are joined together through proline. Such compounds exhibit varying degrees of phagocytosis or pinocytosis stimulation or inhibition.

BACKGROUND OF INVENTION This invention relates to novel, therapeutically useful polypeptides which exhibit varying degrees of phagocytosis or pinocytosis stimulation or inhibition.

The gamma globulin fraction of whole mammalian blood is the fraction which contains the antibodies utilized by the body in resisting invasion by antigens. More specifically, the gamma globulin fraction of mammalian blood is the fraction containing substance which the body utilizes in combatting attack by infectious diseases. The production of antibodies is a natural defense mechanism of the body stimulated by the presence of antigens in the body. Normally specific antibodies are produced to combat specific antigens and, in many instances the body thereafter maintains an antibody level against the specific antigen or infectious organism so that reinfection is inhibited and often prevented.

The use of the gamma globulin fraction of mammalian blood as a therapetutic agent has therefore attracted considerable medical attention since it would seem possible to utilize this fraction from an individual who has successfully overcome an infection to stimulate resistance to that same infection in another individual.

Unfortunately, this approach to prophylaxis has not proved sufliciently fruitful and cannot be used on a long term basis except in cases of a gamma globulinemia. There are many reasons for this. One is that patients often reject gamma globulin, especially on repeated dosages because they treat the gamma globulin as an antigen and develop antibodies to reject it. Another is that an increase above the normal gamma globulin level in the blood may have untoward effects such as seen in hyper gamma globulinemia. Moreover, even in those instances where gamma globulin treatment can be employed the treatment is not as effective as desired because the bulk of it tends to stay in the blood of the patients rather than diffuse into the tissues which is the usual situs of infection.

It has now been discovered that a tetrapeptide L-threonyl-L-lysyl-L-prolyl-L-arginine, which can be isolated from gamma globulin by procedures to be described hereinafter, but which does not exist as a discrete and free molecule in gamma globulin, has the ability to stimulate phagocytosis and subsequent destruction of bacteria by blood polymorphonuclear leucocytes especially neutrophilic leucocytes in mammals. It also stimulates pinocytosis to the same extent allowing the cells to obtain nourishment from the surrounding medium. This application is principally concerned with the class of polypeptides of which this tetrapeptide is one member, with analogs, derivatives and pharmacologically acceptable salts thereof.

THE INVENTION 1 A new class of therapeutically useful polypeptides has been discovered. The members of the class are useful for their phagocytosis or pinocytosis stimulating or inhibiting activity. The principal characteristics of the class are that 'Arg-Pro-Arg-Thr its members are non-antigenic, contain at least three and up to about nineteen amino acids, and contain at least one peptide unit in which two basic amino acids are joined together by one or two proline molecules. The lowest member of the class therefore is a tripeptide in which two basic amino acids are linked through proline.

For convenience in describing this invention the conventional abbreviations for the various amino acids will be used. They are all familiar to those skilled in the art, but for clarity the most important of those with which this invention is concerned are listed below:

Ice

LysineLys Serine-Ser ArginineArg TyrosineTyr Ornithine-Orn Pheny1alaninePhe HistidineI-Iis ProlinePro Threonine-Thr The first four of these acids are the basic amino acids, and they are included within the term basic amino acids as used in this specification and the appended claims.

The basic unit of the class of polypeptides with which this invention is concerned is a tripeptide in which two basic amino acids are joined through proline. Thus, the

following tripeptides may be considered. as within the class:

Lys-Pro-Lys Orn-Pro-Lys Arg-Pro-Arg His-Pro-His Arg-Pro-Lys His-Pro-Lys Lys-Pro-Arg His-Pro-Arg Arg-Pro-Orn His-Pro-Orn Orn-Pro-Arg Lys-Pro-His Orn-Pro-Orn Arg-Pro-His Lys-Pro-Orn Orn-Pro-His Thr-Lys-Pro-Arg Thr-Arg-Pro-Orn Ser-Lys-Pro-Arg Thr-Lys-Pro-Orn Thr-Lys-Pro-Lys Thr-Lys-Pro-His Thr-Arg-Pro-Arg Ser-Lys-Pro-His Thr-Arg-Pro-Lys These compounds in which the amino acids are all in the L-form are preferred as phagocytosis and pinocytosis stimulators. All appear to be more active than the corresponding tripeptides or any of the tripeptides mentioned above. The first mentioned tetrapeptide is by far the most active. The others are somewhat less active. The activity can be further decreased by replacing at least one of the L-form amino acids with a D-amino acid. In fact it may be possible to convert the stimulators into inhibitors by such changes.

One very active inhibitor or antagonist is the reverse isomer of the first mentioned tetrapeptide in which all of the amino acids are in the D-form. This compound is:

Arg-Pro-Lys-Thr Various degrees of inhibition can be achieved by replacing one or more of the D-amino acids with L-amino acids. The following compounds therefore are within the scope of the invention: a v Arg-Pro-Lys-Ser Orn-Pro-Arg-Thr Lys-Pro-Lys-Thr Orn-Pro-Lys-Thr His Pro-Lys-Thr Lys-Pro-Arg-Thr His-Pro-Lys-Ser In general, the most active stimulators are found amongst those tetrapeptides in which all of the amino acids are in the L-form, and the fourth amino acid joined to the basic tetrapeptide unit is an hydroxyl substituted, aliphatic amino acid joined through its carboxyl group to the alpha amino group of the basic amino acid, the carboxyl group of which is joined to the proline in a peptide bond. The reverse position isomers of these compounds in which all of the amino acids are in the D-form are active inhibitors.

Pentapeptides in which one of the amino acids is repeated also have an inhibitory effect, and are therefore useful therapeutic agents. Thus the following compounds are included with the scope of the invention:

Thr-Lys-ProPro-Arg Thr-Lys-Pro-Arg-Arg Thr-Thr-Lys-Pro-Arg Ser-Lys-Pro-Pro-Arg Thr-Lys-Lys-Pro-Arg These structures may also be the repeating unit in polypeptides within the scope of the inveniton containing up to about nineteen amino acids.

In this application, unless the amino acid is specifically identified as D or L, it should be understood that both forms are intended.

It is apparent then, that it is possible to prepare and utilize compounds within the defined class which will have varying degrees of stimulating or inhibiting activity. This is most important because there are medical syndromes in which the patient is substantially incapable of phagocytosis or pinocytosis, as in the case of the splenectomized individual, and conditions where the phatgocytes are so active that other apparently normal cells are ingested. This occurs with patients afilicted with the so called collagen diseases such as rheumatoid arthritis and lupus erythematosus. Abnormal phagocytic activity in these patients may well be the cause of the destructive vascular lesions in the joints and various organs. Treatment with the inhibitory compounds of this invention is indicated in these patients.

As stated above, the presently preferred compounds are tetrapeptides. Of these, the compound with the highest order of stimulating activity yet noted is Thr-Lys-Pro- Arg in which all of the amino acids are in the L-form. The preferred inhibitor is Arg-Pro-Lys-Thr in which all of the amino acids are in the D-form. For convenience the first named compound has been called tuftsin IV and the second named compound is called retrotuftsin IV.

The invention will now be described more specifically by reference to tuftsin This tetrapeptide has been synthesized chemically and shown to be identical with one of the products which can be isolated in extremely minute quantities from gamma globulin by very tedious procedures which involve its cleavage from leukokinin, one of the known protein fractions in gamma globulin. Leukokinin is synthesized or activated in the spleen. splenectomized individuals, therefore, are deficient in tuftsin IV.

Tuftsin IV as it exists in the blood of mammals is a part of the larger molecule leukokinin, as is clear from its method of isolation which requires a specific enzyme leukokininase for its cleavage. It appears to stimulate phagocytosis and pinocytosis by a mechanism which involves (a) binding of leukokinin at a specific site on the cell membrane, (b) cleavage from the leukokinin molecule through the action of the membrane enzyme leukokininase which forms an integral part of the binding site, and (c) transport as a complex with the enzyme to the site of its activity where it is apparently destroyed as its discharges its stimulatory action. In any event, no procedure is known which does not require enzymatic cleavage and which will permit the isolation of the pure tetrapeptide. In fact, no procedure is known which will permit the isolation of useful quantities of tuftsin IV from any practical quantity of mammalian blood.

Leukokinin itself is not useful as a therapeutic agent s w v because the molecule cannot be obtained as an autologous molecule, but only from pooled heterologous sources. Because of this and its large size and differing structure it triggers an antigenantibody reaction in the host and is rejected. Tuftsin IV, however, appears to be identical in all human leukokinin and, because it is a small molecule, it does not cause an antibody response.

Moreover, because it is a small molecule it is readily transported into the tissues where it elicits a phagocytosis effect on any organism or particle through its direct action on the phagocyte and not on the bacteria. In this sense tuftsin IV functions as a general antibiotic, but unlike antibiotics, it does not evoke an allergenic response nor does it give rise to the emergence of resistant organisms as so often happens in bacterial infections treated with antibiotics that act selectively on the bacetria thereby allowing resistant mutants to survice. Tuftsin IV is a non-oxic, non-antigenic, therapeutically useful, phagocytosis and pinocytosis stimulatnig tetrapeptide which arouses the bodys own natural defenses against invasion by the foreign substance.

Tuftsin IV and related polypeptides, tuftsin I, tuftsin H and tuftsin III, all of which are phagocytic and pinocytic, may be obtained from various fractions of gamma globulin that have been chromatographed on phosphocellulose columns. The general procedure is outlined below.

Gamma globulin is obtained from human, dog or rabbit sera by precipitation at 0.33 ammonium sulfate saturation. The procedure for the isolation of the tuftsins is illustrated in detail in the examples. Tuftsin I is obtained by treatment of a fraction from a phosphocellulose column (PCI) with trypsin or enzymes obtained from the membranes of red blood cells and the membranes of polymorphonuclear neutrophilic white blood cells.

Tuftsin II, HI and IV are obtained from phospocellulose fraction IV (PC IV) by treatment of this fraction with trypsin or the enzyme leukokininase which is prepared from the membranes of blood neutrophilic leucocytes and from rabbit peritoneal exudates by known procedures. They are principally peptides and are separated from the balance of the protein, after the enzyme treatment, by precipitation of the latter with 5% trichloroacetic acid. The supernatant contains the tuftsin compounds. Trichloracetic acid is removed by ether extraction and the tuftsins are lyophilized and chromatographed on Sephadex G-10 columns equilibrated with a running eluting fluid composed of 0.1 N acetic acid saturated with chloroform. The effluent is collected in 1 ml. samples. Samples to contain tuftsin II, and samples to 158 contain tuftsin III and IV. The effluent containing tuftsin III and IV is lyophilized and taken up in pyridineacetate buffer at pH 4. The mixture is chromatographed on Dowex 50 columns and eluted at pH 4.0 to 6.0 in 1.2 to 2.5 M pyridine concentration (linear gradient technique). The eflluent is collected in 2 ml. samples. Tuftsin HI in samples 20 to 35, and tuftsin IV is in samples 65 t0 Tuftsin IV when subjected to thin layer chromatography utilizing 2:1 propanol-water yielded a single band. Dansylation of the peptide with dansyl chloride results in a single fluorescent band on thin layer chromatography with silica gel using 15:4:1 chloroform-methanel-acetic acid.

Hydrolysis of tuftsin IV with 6 N HCl at 105 C. for 20 hours yields threonine, lysine, proline and arginine in the molar ratio 1.00:1.00:1.05:0.92. With carboxypeptidease B digestion, only arginine is released as the carboxy terminal amino acid. Following digestion with leucine aminopeptidase, only threonine is released as the amino terminal acid. Furthermore the products of the enzymatic reactions when dansylated yield only threonine and arginine as the free dansyl components. Positive Sakaguchi staining further identifies, on silica gel thin layer, the presence of arginine as the carboxy terminal amino acid after reaction with carboxypeptidase B. Tritium exchange following treatment of the peptide with acetic acid an hydride in pyridine in tritium water established arginine as the only radioactive residue. The presence of proline between lysine and arginine is established by the resistance of the peptide to trypsin digestion. After removal of arginine by carboxypeptidase B, refluxing of the remaining peptide with acetic acid anhydride for 45 minutes in tritium water showed proline as the only radioactive residue. Dansylation of the peptide after removal of threenine as the free dansyl components. Positive Sakaguchi the next amino terminal. Thus the structure of tuftsin IV is established as Thr-Lys-Pro-Arg. This is further confirmed by sequential Edman degradation and the identification of the thiohydantoin residues as well as by dansylation of the exposed residue and the Edman subtractive method. The identity of tuftsin IV is ultimately established by its synthesis from the component amino acids as outlined below and illustrated in the examples.

By similar procedure, the structure of tuftsin I, II and HI is established as principally polypeptide in nature.

Tuftsin IV can also be isolated from commercially available Cohn fraction II gamma globulin by the process illustrated in the examples, but not in practically useful qualities.

The phagocytic activity of the products of this invention is established by the following test:

Buffy (white cell) coats from dog or human blood or peritoneal exudate from rabbit or guinea pigs were prepared, washed three times with Krebs-Ringer medium and suspended in the same medium. A otal of 0.15 ml. of this medium containing 45 10 neutrophilic leucocytes per cubic millimeter were pipetted into a reaction vessel. Next 0.1 ml. of the tuftsin mixture containing 0.2 to 2 were added to the vessel followed by 0.05 ml. of staphylococci opsonized in inactivated opsonin rich serum. This represented 1.5 to 2 bacteria per neutrophile. The final volume was 0.3 ml. This was rotated at eight cycles per minute at 35 C. for /2 hour. Smears were made and stained with Wrights stain. The number of cells containing engulfed bacteria for each 100 cells counted is the index of phagocytosis.

The stimulatory eifect of tuftsin IV and the related products of this invention is destroyed by incubation for 3 hours at 30 C. with pronase, subtilisin, carboxypeptidase B and leucine aminopeptidase, using 40 ,ug. of protein per ml. under optimum conditions of enzyme action. Similarly it is destroyed by incubation with soluble supernatants of disrupted leucocytes and membrane preparations from human erythrocytes and rat liver at pH 7.0 in phosphate bulfer. In all of these media phagocytosis is at the same level as the controls. Tuftsin 1V is not destroyed by exposure to aeration in the presence or absence of cysteine, heating at 80 C. for minutes in 80% ethanol, enzymatic treatment with trypsin, chymotrypsin, clostridiopeptidase B, phosphatase, deoxyribonuclease or ribonuclease. Phagocytosis levels obtained after such treatments are about 2.5 times those of the controls. Tuftsin IV is soluble in 95% alcohol, pyridine and acetic acid and insoluble in ether.

The products of this invention are useful mammalian therapeutic agents and are effective as stimulating or inhibiting agents at extremely low levels. The physician or veterinarian will determine the dosage which will be most suitable for a particular application. It may vary from patient to patient depending on the size of the patient, the condition under treatment and other factors which are readily evaluated by those skilled in the art. For continuous administration over extended periods to individuals with more or less permanent metabolic abnormalities or splenectomized individuals the products will normally be provided in various dosage forms varying from relatively large to build up a prompt blood level to relatively small to maintain an effective level. For intermittent treatments to combat acute or chronic infections various dosage forms may be provided. The products maybe administered at very high levels, even up to two or more grams per day. Normally they will be provided in dosage units containing about 250 mg. of the active ingredient and the number of units appropriate to the condition under treatment can be prescribed per day.

The products of this invention may be administered alone but will generally be administered with pharmaceutically acceptable, non-toxic carriers, the proportions of which are determined by the suitability and chemical nature of the particular carrier, the chosen route of administration, and standard pharmaceutical practice. For example, in combatting various infections or in maintaining therapeutically elfective levels in the blood or tissues may be administered orally in the form of tablets or capsules containing such excipients as starch, milk sugar, certain types of clay, etc. They may be enteric coated so as to be more resistant to the acid With digestive enzymes of the stomach. For intravenous and intramuscular administration they may be used in the form of a sterile solution containing other solutes, for example, enough saline or glucose to make the solution isotonic.

It is a particular advantage of the products of this invention that unlike many peptide bond containing therapeutic products they can be administered orally because they are resistant to enzymatic hydrolysis by the enzymes of the lower digestive tract. Because of their amphoteric nature they may be adsorbed for oral administration on non-toxic ion exchange resins which may be either anionic or cationic to achieve slow release either in the stomach or the intestines or both. Furthermore, adsorption on these resins makes them all the more resistant to enzyme destruction.

Another advantage arising from the amphoteric nature of the products of this invention is that they can be utilized in the form of pharmacologically acceptable salts which may be either metallic salts or acid addition salts. These salts have the advantage of water solubility and are particularly useful for parenteral administration. The metallic salts, especially the alkail metal salts are relatively stable and for that reason are preferred over acid addition salts. The sodium salts are especially preferred because of their ease of preparation.

The acids which may be used to prepare the pharmacologically acceptable acid addition salts of this invention are those containing non-toxic anions and include, for example, hydrochloric, sulfuric, phosphoric, acetic, lactic, citric, tartaric, oxalic, succinic, maleic, gluconic, saccharic, and the like acids.

The polypeptides of this invention can be synthesized by any of a wide variety of techniques now available for the synthesis of simple and complex polypeptides and even relatively low molecular Weight proteins. In general, these techniques involve stepwise synthesis by successive additions of amino acids to produce progressively larger molecules. The amino acids are linked together by condensation between the carboxyl group of one amino acid and the amino group of another amino acid to form a peptide bond. In order to control these reactions it is necessary to block the amino group of the one acid and the carboxyl group of the other. Necessarily, the blocking groups must be easily removed. The whole series of reactions must take place without causing racemization of the products. Certain amino acids have additional functional groups, for example, the hydroxyl group of tyrosine or'of threonine. It is usually necessary to block these additional groups with an easily removed blocking agent so that it does not interfere with the condensation reaction.

A large number of procedures have been devised by the art for the synthesis of polypeptides and a wide variety of blocking agents has been devised. Most of these procedures are applicable to the synthesis of the class of polypeptides to which this invention pertains. No useful purpose would be served by describing the application of all of them although several are specifically illustrated in the examples.

The presently preferred procedures for the synthesis of the polypeptides of this invention are the Merrifield technique and the N-carboxy anhydride technique. In the former an amino acid is first bound to a resin particle, as by an ester bond and the peptide is generated in a stepwise manner by successive additions of protected amino acids to the growing chain. In the latter, an N-carboxyl amino acid anhydride is reacted with the amino group of a second amino acid or peptide under conditions such that the only amino group present in appreciable concentration in reactive form during the course of the reaction is the amino group which is to participate in the reaction. This control is effected by selection of concentration, temperature, time and hydrogen ion concentration. The coupling reaction normally takes place under alkaline conditions, usually at a pH of from about 8.5 to 11. The intermediate carbamate is then decarboxylated by lowering the pH to from about 3 to 5. The product formed may be reacted with another N-carboxy amino acid anhydride without isolation and under substantially the same conditions. The process affords a very rapid method for the production of polypeptides.

Tuftsin IV and retrotuftsin IV are both synthesized by these and other techniques as illustrated in the examples. Tuftsin IV as prepared synthetically has proved to be identical with the product isolated by the procedure described above.

One of the reasons tuftsin IV and retrotuftsin IV are preferred members of the class is their high order of activity. Another is because they lend themselves so readily to slight molecular modifications to produce compounds which have more or less activity than the parent tetrapeptide; or may be more or less readily absorbed into the tissues; or particularly suitable for a selected route of administration.

While tetrapeptides are the preferred members of the class, the compounds with which this invention is concerned may be generally described as therapeutically useful, non-antigenic polypeptides containing at least three and up to about nineteen amino acids comprising one unit or repeating units, each unit containing three to five amino acids, each repeating unit being joined to an adjacent unit by proline or cystine, each unit containing two basic amino acids linked together by one to two proline molecules. Although the repeating units will normally be identical, it is not essential that they are. The invention also includes the pharmacologically acceptable salts and derivatives of these polypeptides. Typical members of the class could include therefore:

Lys-Pro-Arg Lys-Pro-Arg-Pro-Lys-Pro-Arg Arg-Pro-Lys-Thr-Cys-Cys-Thr-Lys-Pro-Arg Thr-Lys-Pro-Arg-Pro-Thr-Lys-Pro-Arg-Pro-'Ihr-Lys- Pro-Arg-Pro-'I'hr-Lys-Pro-Arg.

Among the polypeptides containing more than four amino acids, the nonapeptides are preferred because they are not too difficult to prepare. They are especially useful for those patients who are on a continuous regimen of tuftsin therapy since they can be used to decrease the number of dosage units which must be ingested without concurrent decrease in blood levels. The molecular weight of the nonapeptide is so low that there is substantially no danger of an antigenic or allergenic response even in hypersensitive individuals. Furthermore, because of the low molecular weight the nonapeptide readily diffuses through the peritoneum.

While, at least in theory, almost any amino acid can be used to link the repeating units, the preferred linking amino acids are proline and cystine. The reason for this is that these amino acids form peptide bonds which are relatively resistant to the enzymes of the digestive tract. Since the high molecular weight products of the invention will normally be used by patients under long term treatment it is preferred that the therapeutic agent be ingested orally. It is essential, therefore, that the oral agent is not one which will be sufiiciently resistant to digestion to permit absorption.

Any of a wide variety of non-toxic derivatives of the polypeptides of this invention can be usefully employed. The pharmacologically acceptable salts have been mentioned above. Amides, esters, acylated derivatives and others can also be utilized.

Tuftsin IV, for example, can readily be obtained as an amide. For example, the tetrapeptide can be reacted with thionyl chloride to form the acid chloride, and the latter reacted with ammonia under conditions that minimize racemization to form the amide. Alternatively, the tetrapeptide can be formed by joining proline, lysine and threonine successively to an arginine ester and then converting the resulting tetrapeptide ester to an amide by ammonolysis. The ester may be, for example, any lower alkanol ester containing up to about six carbon atoms.

Enzymes are available in the body which will hydrolyze both amide and ester groups to regenerate the stimulatory activity of the acid. Both ester and amide derivatives are useful therapeutic agents because of their increased chemical stability compared with the free acids. They have altered rates of absorption or diffusion into the tissues and delayed excretion through the kidneys. They may be used in the form of pharmacologically acceptable salts.

Other useful derivatives may be obtained by modifying the free functional groups on the polypeptide backbone, for example, free hydroxyl groups or free amino groups. One very convenient class of derivatives is the class in which a free hydroxyl group, for example, the free hydroxyl group of threonine or tyrosine is esterified with an alkanoyl or alkenoyl group containing up to eighteen or more carbon atoms. Alternatively, an amino group, for example, the amino group of threonine or lysine can be acylated with an alkanoyl or alkenoyl group containing up to about eighteen carbon atoms. In both instances the preferred derivatives are those in which the derivatizing groups contain from eleven to eighteen carbon atoms because the longer hydrocarbon chains impart increased lipid solubility to the molecules and enhance their transport across cell barriers.

Both types of derivatives may be prepared directly from the polypeptide, but are preferably prepared by incorporating in the peptide during synthesis of an amino acid with the selected group, for example, the alkanoyl group already in place.

While the tuftsins and other polypeptides of this invention are, in a sense, natural therapeutic agents and can be used alone for that purpose, they will often be administered together with one or more other therapeutically active materials such as an antibiotic, antifungal or antiviral agent. One reason for this is to combat acute, potentially lethal infections with all of the resources available. Another is to clean up the toxins, especially the endotoxins of gram negative bacteria and other debris which accumulate in the tissue and the blood as a result of the death of infectious microorganisms. The toxemia resulting from such accumulations is sometimes as dangerous, if not more so, to the health of the patient as the original infection. The presence of tuftsin IV or its analogs helps the body eliminate the endotoxins. The products of the invention may be coadministered with such materials as tetracycline, chlortetracycline, neomycin, erythromycin, novobiocin, penicillin, chloramphenicol, and nitrofurazone.

It is also possible to chemically combine the compounds with other agents such as antibiotics in order to bring both materials to the same site simultaneously so that they can both exert their salutory effects. For example, the carboxyl group of penicillin can be used to esterify tuftsin IV through the hydroxyl group of threonine. It can also form an amide linkage through the alphaamino group of threonine or the epsilon amino group of lysine;

The stimulatory peptides of this invention may also be used to preserve the usefulvalues of whole blood. It has been observed that on standing, even under normal refrigeration, whole blood loses its phagocytic activity and the white cells start to disappear. The addition of small amounts of, for example, tuftsin IV will reverse this trend.

The following non-limiting examples are given by way of illustration only.

EXAMPLE 1 Tuftsins from gamma globulin through leucokinin Gamma globulin (150 mg.) from Cohn fraction II, in 10 ml. of 0.05M acetate buifer (pH 4.8) was applied to a cellulose phosphate column (4 x 12 cm.) previously equilibrated with the same buffer. The flow rate was adjusted to 2 ml./min. and the efiluent was collected in 4.0 ml. portions. Three to four holdup volumes of the solvent bulfer (400 to 550 ml.) were run through the column to ensure thorough escape of the occasional contaminating albumin. No gamma globulin appears in this efiluent. The first gamma globulin fraction was eluted with 0.05 M acetate buffer (pH 4.8) in 0.15 M NaCl and contained 51 mg. of protein (34%). Elution with the same buffer was continued until the absorbance at 280 m corresponded to that of the buffer. This was routinely achieved with each fraction after 1.3 to 1.7 holdup volumes of the eluting butter had passed through the column, i.e. until 180 to 230 ml. of efiluent was collected. The second fraction contained 37 ml. of gamma globulin (25.0%) and was eluted using 0.05 M acetate buifer (pH 5.0) in 0.15 M NaCl; and the third fraction, 29 mg. (19%), was eluted at pH 5.2 under otherwise identical concentrations of buffer and salt concentration. Finally the fourth fraction, 20 mg. of gamma globulin (13%) was eluted by raising both the pH to 5.4 and the NaCl concentration to 0.2 M. Analysis of the results in terms of total protein recovered in each fraction gave a standard deviation of around i5% for the first two fractions and around :L-2% for the last two.

Rechromatography was then performed on the separate fractions. Each fraction was adjusted to 0.60 saturation with solid ammonium sulfate and allowed to stand for 8 hours at The precipitated protein was then collected by centrifugation and dialyzed against 0.05 M acetate buffer at pH 4.8. Conditions for rechromatography were as detailed above. This method of preparation, presented above, was used to accumulate a few 100 mg. of fraction IV (PC IV) containing leukokinin.

100 mg. of fraction PC IV were digested with leukokininase prepared from human, dog or rabbit polymorphonuclear leucocyte membranes. pg. of leukokininase protein were added per mg. of PC IV, in a final volume of 25 ml. of 0.1 M phosphate buffer pH 6.7. This was incubated for two hours at 37 C. The reaction was stopped by adding trichloroacetic acid to a final concentration of 5%. The precipitate was separated out. After extraction with ether to remove the trichloroacetic acid, the material was lyophilized and fractionated on Dowex 100 column equilibrated with the eluting fluid composed of 0.1 M acetic acid and saturated with chloroform; column bed volume 295 ml.; void volume 1l5ml.; height 118 cm.; inside diameter 1.8 cm. 1 ml. samples were collected in tubes in an automatic fraction collector. The eflluent in tubes No. 115-125 contain tuftsin II and the effluent in tubes No. 135 to 158 contain tuftsin IH and IV. The efiluent containing tuftsin III and IV was lyophilized and taken up into pyridine-acetate buifer pH 4 containing 1.2 M pyridine. This was then chromatographed on aminex columns; height 60 cm.; internal diameter 0.6 cm.; and eluted with a linear gradient at 60 C. The starting buffer is pyridineacetate pH 4.0 containing 1.2 M pyridine; and the final butter pyridine acetatepH 6.0 containing 2.5 M pyridine.

2 ml. samples were collected in each tube. The efiluent in tubes No. 20 to 35 represented tuftsin III. Tuftsin IV was collected in the effluent of tubes No. 65 to 78. This latter represented a sharp symmetrical peak. On thin layer chromatography with silica gel, using a developing solution with a ratio of propanol 2, water 1, it yielded one single band. Dansylation of the peptide with dansylchloride also showed one single fluorescent band on thin layer chromatography on silica gel using a developing solution with a ratio of chloroform 15, methanol 4 and acetic acid 1.

The amino acid composition after hydrolysis in 6 N HCl at 105 C. for 20 hours, yielded molar ratios of threonine 1.01, lysine 1.00, proline 1.07, and arginine 0.90. All amino acids were in the L-form. With carboxypeptidase B digestion with per ml. at 20 for 2 hours pH 7.4, only arginine was released as the carboxy-terminal and at the level of 50% of the peptide. Following digestion with leucine aminopeptidase with 80' per ml. at 25 for 2 hours at pH 8.5 with MgC1 3 10- M, only threonine was released as the aminoterminal to the extent of 30% of the peptide. The sequence proved to be Thr-Lys-Pro- Arg, and the amount recovered was about 1,000 g. giving a yield of 1.0 g. per 1,000 g. of fraction PC IV or 1.0 pg. per 10,000 g. of whole gamma globulin.

EXAMPLE II Tuftsins from Cohn fraction II 1 g. of Cohn fraction II gamma globulin was digested with trypsin, 5 mg. per mg. of the gamma globulin in tris hydrochloride bufier 0.1 M containing calcium 0.01 M pH 8.1 for 1 hour at 37 C. final volume 250 ml. The released tuftsin was separated from the rest of the gamma globulin by precipitation in 5% trichloracetic acid and the precipitate discarded. The supernatant was extracted three times with ether and the material lyophilized, taken up in 0.5 ml. of 0.1 M acetic acid and chromatographed on Sephadex G-l0 columns, equilibrated with 0.1 M acetic acid which also served as the eluting solution; column bed volume 295 ml.; void volume ml., height 116 cm., inside diameter 1.8 cm. 1 ml. samples were collected in tubes in an automatic fraction collector. The efiluent in tubes 115 to contains tuftsin H and the efiluent in tubes and 158 contains tuftsin III and IV. The eflluent containing tuftsin III and IV was lyophilized and taken up into pyridine-acetate buffer pH 4 containing 1.2 M pyridine. It was then chromatogrphed on aminex columns, height 60 cm., internal diameter 0.6 cm., and eltued with a linear gradient at 60 C. The starting bufier was pyridine-acetate pH 4.0 containing 1.2 M pyridine; and the final buflfer pyridine-acetate pH 6.0 containing 2.5 M pyridine. 2 ml. samples were collected in each tube. The effluent collected in tubes 20 to 35 represented tuftsin III. Tuftsin IV was collected in the effluent of tubes 65 to 78. This latter represented a sharp symmetrical peak. On thin layer chromatography with silica gel, using propanol 2, water 1, it yielded one single band. Dansylation of the peptide with dansylchloride also showed one single fluorescent band on thin layer chromatography with silica gel using chloroform 15, methanol 4 and acetic acid 1.

The amino acid composition after hydrolysis in 6 N HCl at 105 C. for 20 hours, yielded molar ratios of threonine 1.03, lysine 1.00, pyroline 1.04 and arginine 0.93. With carboxypeptidase B'digestion under conditions described in Example I, only arginine was released as the carboxy-terminal and at the level of about 60% of the peptide. Following digestion with leucine aminopeptidase as in Example 1, only threonine was released as the aminoterminal to the extent of 35% of the peptide. All amino acids were in the L-form. Y

, EXAMPLE HI Tuftsin IV by direct trypsin digestion of gamma globulin Fresh gamma globulin was prepared from 100 ml. of human or dog serum as follows: to 100 ml. of serum were added 100 ml. of 0.15 N sodium chloride and 100 ml. of saturated ammonium sulphate solution. The precipitate was allowed to form at room temperature for two hours. It was centrifuged down and the precipitate dissolved in 20 ml. of 0.15 N sodium chloride. To this were added 10 ml. of saturated ammonium sulphate. After two hours of standing at room temperature it was left at 5 C. overnight. The preparation was then centrifuged down and the supernatant discarded. The gamma globulin precipitate was then dialyzed against 1 liter of 0.15 N sodium chloride for 4 hours. The dialysis was repeated 2 more times.

1 g. of the freshly prepared gamma globulin was then digested with trypsin and processed on Dowex and aminex columns exactly as in Example II. The results of the analysis of tuftsin IV were also similar, threonine 0.98, lysine 1.0, proline 1.03, and arginine 0.87.

EXAMPLE IV Tuftsin IV from freshly prepared gamma globulin Freshly prepared gamma globulin, as in Example III, Was fractionated on phosphocellulose columns as in Example I. 100 mg. of fraction IV were then treated with leukokinase and fractionated on Dowex 100 and aminex columns, as in Example I. The isolated tuftsin IV gave identical residues with similar values as in Examples I, II, and IH, threonine 0.92, lysine 1.00, proline 1.04 and arginine 0.97.

EXAMPLE V Synthesis of tuftsin IV t-BOC-N -nitroarginine, 13.8 mmoles were reacted with 10 g. of chloromethyl-resin (polystyrene divinyl benzene chloromethyl resin 2.2 mmoles of chloride per gram of resin) in a mixture of triethylamine, 12.4 mmoles, and 30 ml. ethanol for 24 hours at 80 C. with constant magnetic stirring. The resin was then thoroughly washed with acetic acid, then with absolute ethanol, water with increasing concentration of ethanol, finally with absolute ethanol followed by methanol and methylene chloride. The resin was dried in vacuo to constant weight. 30 mg. of the resin was hydrolyzed with 6 N HCl in dioxane for 24 hours. The filtrate was dried in vacuo and dissolved in citrate buifer pH 2.2, 0.2 M. A proper aliquot was assayed in the amino acid analyzer. Ornithine, arginine and nitroarginine were measured. The first two are by-products of the acid hydrolysis of nitroarginine-resin. The summation of all three represents the amount of esterified t-BOC- nitro arginine which amounted to 0.3 mmoles per gram of resin.

1.5 g. of resin representing 0.45 mmoles to t-BOC-nitroarginine were placed in the Merrified solid phase vessel secured onto a clamp to the shaft of a 180 reversible stroke motor. All manipulations henceforth take place at room temperature with 180 rocking motion such that the resin is constantly agitated. The protecting t-BOC group was cleaved with ml. of 50% of trifluoroacetic acid (TFA) in methylene chloride for a 30 minute reaction time. The resin was then Washed with methylene chloride followed by chloroform 3X each with 15 ml. 1 ml. triethylamine plus 9 ml. of chloroform were added to the resin and shaken for 10 minutes to neutralize the hydrochloride. This was washed again with chloroform and methylene chloride as before. t-BOX proline, 1.35 mmoles in 15 ml. of methylene chloride, was then coupled to the N of nitroarginine with 1.35 mmoles of dicyclohexylcarbodiimide (DCC) for two hours with continuous shaking. The resin was washed with ethanol, chloroform and methylene chloride, 15 ml. of each 3 times, respectively. The t-BOC group was removed from the proline with 50% trifiuoroacetic acid in methylene chloride, washed and neutralized with triethylamine as before. In a similar manner N-t-BOC N carbobenzoxy (CBZ) lysine 1.35 mmoles was coupled to proline, deprotected, neutralized and 1.35 mmoles of N-t-BOC-O-benzyl threonine was then coupled to the deprotected N of lysine. Thus the steps of deprotection (cleavage of the t-BOC alone) with TFA, washing, neutralization with triethylamine, washing and coupling with DCC was repeated for each amino acid residue in an identical manner.

30 mg. of the tetrapeptide resin were hydrolyzed with 6 N hydrochloric acid in dioxane for 18 hours and the product assayed on the amino acid analyzer. 'It yielded a ratio of Thr 0.9, Lys 1.0, Pro 1.08, Arg 1.0. The value for arginine is again the sum of arginine, ornithine and nitroarginine. The tetrapeptide was cleaved off the resin with hydrogen bromide in TFA at room temperature for 1 /2 hours. It was then dried in vacuo, washed twice with water and lyophilized. Its weight of 176 mg. represented a 78% yield based on arginine esterified. It was then taken up in 10 ml. of methanol containing 10% acetic acid. This was then exposed to catalytic hydrogenation with twice its weight 350 mg. of palladium on barium sulfate in a hydrogen atmosphere at pounds per square inch pressure with continuous shaking for 24 hours at which time no nitroarginine was detected at 271 mp, the peak of absorption. An aliquot was again hydrolyzed with 6 N HCl in water and assayed. This yielded a comparable figure, Thr 0.92, Lys 1.0, Pro 1.05, Arg 0.98.

The material was then chromatographed on an aminex column with a linear pH gradient 46 with pyridine 1.2 M to 2.5 M at flow rate of 8 ml. per hour. The major peak, the tetrapeptide, appeared exactly at the tuftsin IV peak derived from CP fraction 1V gamma globulin, i.e. between 138-150 ml. with a peak with 144 ml. of effiuent. The collected material was dried in vacuo, washed twice with 0.1 molar acetic acid, taken up in 0.1 molar acetic acid, and lyophilized. Upon amino acid assay a ratio of Thr 0.96, Lys 1.0, Pro 1.03, Arg 1.0 was observed. All amino acids were in the L-form.

The tetrapeptide which was in the .form of the diacetate salt was then converted to the sodium salt by the addition of three equivalents of sodium hydroxide.

The final yield based on the nitroarginine esterified to the resin was about The process was repeated to form the tetrapeptide with all amino acids in the D-form. It was repeated twice with serine in place of threonine. Both all L- and all D-forms were prepared.

EXAMPLE VI Synthesis of tuftsin IV t-BOC-nitroarginine is esterified to the chloromethylated resin, deprotected, neutralized and washed as in Example plus an additional wash with dimethylformamide (DMF). 1.2 g. containing 0.25 mmole of arginine is used as starting material. t-BOC pnoline succinimide ester, 1.0 mmoles is added to the resin in DM-F and allowed to react for 24 hours with shaking at room temperature. Deblocking, neutralization, and washing as in Example V with a final wash with DMF repeated for each succeeding coupling with 1.0 mmole of N hydroxysuccinimide esters of N-t-BOC N-CBZ lysine and N-t-BOC-O-benzyl threonine. The cleavage from the resin, catalytic hydrogenation, column purification are exactly the same as in Example V. The aminex column, as in Example V, showed a predominantly major peak at the tuftsin IV level. The yield based on arginine esterified was 34%. After cleavage from the resin, the ratios were Thr 0.85, Lys 1.0, Pro 0.95, Arg 1.08. After aminex purificatioTl, the ratios were Thr 0.93, Lys 1.0, Pro 1.05, Arg 0.98. The tetrapeptide was dried in vacuo, washed with 0.1 M acetic acid twice and finally lyophilized in 0.1 acetic acid and converted to the sodium salt as in Example V.

The tripeptides Lys-Pro-Arg with all acids in both D- and L-forms were similarly prepared by omitting the reaction with the protected threonine.

13- EXAMPLE VII" I.) NF-t-BOC' N -nitroarginine' was esterified to the resin as described in Example V. It was deprotected, washed 1n'nitralized as in Example V. 1.2 gfcontaining 0.32 mmole of'jarginine' was reacted with 1.28 mmoles of t- BQCsproline-p-nitrophenyl. ester in DMF 10 ml. with shaking for 18 hours at room temperature. When the reaction was completed, .the t-BOC was cleaved, the resin neutralized andwashed as in Example VI. Subsequent couplings were done with N-t-BOC N-CBZ lysine-pnitrophenyl ester followed by-N-tBOC-O-benzyl-threonine-p-nitrophenylester. Depr'otection of the amino groups, washing and neutralization, cleavage .from the resin and catalytic hydrogenation followed as in Example VI. Chromatography on aminex. columns as in Example V was used to isolate pure tuftsin IV. Analysis of the major peak showed Thr 0.90, Lys 1.0, Pro 1.03, Arg 1.03. The final yield of 65% of esterified arginine was obtained.

EXAMPLE VI II Synthesis of tuftsin IV Arginine (monohydrochloride) 0.2 mmole in 2 ml. of water containing 0.13 ml. of triethylamine, was reacted with 0.6 mmole of N t- BOC proline N-hydroxysuccinimide ester ('Osu) vin 5 ml. of tetrahydrofuran with constant stirring at room temperature for 24 hours. It was then dried in vacuo and the residue taken up in 0.1 M acetic acid and lyophilized. The material when then dis- "solved in1water and chromatographed with a linear pyridine acetate gradient on aminex column 0.6 x 60 cm. at 60 C. pH'3.1-5.6, pyridine 0.2 M-4 M, 2 ml. were collected in each tube; fiow rate was 8 ml. per hour. Under these conditions N t-BOC Pro-Osu and the free acid do not bind to the column andappear at the front. The major and first Sakaguchi positive peak representing the dipeptide wasisolated and lyophilized, taken up in water and lyophilized again to constant weight. The protected dipeptide (60 mg.) was then placed in ice bath and 1.0 ml. of ice cold TFA added. The dissolved peptide was kept in the ice bath for minutes. It was then diluted with 40 ml. with ice: cold'wat er and extracted twice with 80 ml.

of ice chilled ether. The combined etherlayers were back extracted with ice water, m 1., two times and all the aqueous; phase pooled lyophilized. The dipeptide was --then taken up, in 2 ml. of, water, 0.12 'ml. of triethylamine was added and reacted with 0.58 mmole of N t-BOC -CBZ lysine-Osu ester in 5 ml. of tetrahydrofuran at room temperature for 24 hours with stirring. The material was again dried in vacuo, taken up in 0.1 M acetic acid and lyophilized. It was then dissolved in water and chromatographed on the same aminex column except that the pyridine acetate "gradient was from pH 4-6 and pyridine 1.' 2 M2 .5 M. .The uhreacted protected lysine-Osu did not bind. The major peak proved to be the tripeptide, Lys- Pro-Arg, 1.0,- 1.08, -0.97,; respectively, on amino acid analysis. It was then'dried and lyophilized as an aqueous solution, deprotected'with'I'FA. and extracted with ether under identical conditions described. above for the dipep- 14 EXAMPLE IX f Synthesis of tuftsin 1v The various steps in the synthesis of the first dipeptide utilizing arginine and N-t-BOC proline p-nitrophenyl ester followed essentially the same procedure as in Example VIII. The time of the reaction was 18 hours. Column fractionation of aminex, deblocking and coupling with N"-t-BOC N-CBZ lysine p-nitrophenyl ester was also as in Example VHI. This was also the case with its purification in aminex, deblo cking and reaction with N-t BOC-O-Benz-threonine-Z,4-dinitrophenyl ester and subsequent purification and isolation of the tetrapeptide and its deblocking with HF followed by the usual chromatography on aminex. It followed essentially the same steps as with the N-hydroxysuccinimide procedure in Example VIII. The yield and purity was also similar to that of the preceding examples with a composition Thr 0.98, Lys 1.0, Pro 1.03, Arg 0.95.

EXAMPLE X Synthesis of tuftsin 'IV Arginine free base 0.5 mmole was dissolved in 0.5 M borate buffer 2.5 ml. pH 10.5 cooled to 0 C. in a rapid mixing small omnimixer kept at 0 C. in an ice bath. 0.55 mmole of the N-carboxy anhydride (NCA) of proline was added in two equal portions 1 minute apart while the pH was maintained by the addition of 10 N sodium hydroxide. One minute after the second addition, the pH was brought down to 4.8 by the addition of concentrated sulfuric acid. The solution was flushed with N gas to drive oil the CO formed from the resulting peptide carbamate. The pH was raised to 10.2 and NCA of N-CBZ lysine 0.55 mmole were added and the reaction allowed to proceed for 2 minutes while maintaining the pH at 10.2 with 10 N NaOH at 0 C. Sulfuric acid was then added to bring the pH down to 5.0 while N gas was passed through. The preparation was then passed through in Sephadex G10 column equilibrated in 0.1 M acetic acid and the peptide isolated, lyophilized and taken up in 5 ml. of tetrahydrofuran containing 0.13 ml. of triethylamine and 1.5 mmoles of N -t-BOc-O-benzyl-threonine. Succinimide ester was added and stirred at room temperature for 24 hours. It was evaporated in vacuo, taken up in water and lyophilized to constant weight. It was then deblocked with TFA at 0 for 20 minutes and the solvent evaporated in vacuo. It was then lyophilized from a water solution and chromatographed in aminex as in Example I. Einal yield 28% in terms of arginine as the starting material. Composition, Thr 0.86, Lys 1.0, Pro 1.03, Arg 0.96.

Retrotuftsin IV was similarly prepared. The analog of tuftsin IV in which tyrosine replaces threonine was similarly prepared. The hydroxyl group of tryosine was protected during synthesis as the tetrahydropyramyl ether in accordance with the process of US. Pat. 3,467,667.

The processes of this example were repeated to prepare the same compounds except that the basic amino acids when used as N-carboxy anhydrides were protected as N-chloromercury salts as described in U.S. Pat. 3,459,760. The yields were much improved.

EXAMPLE XI Formation of acylated tuftsin IV 15 fatty acids with a yield varying from 37% to 67%. This includes acetic, propionic, butyric, lauric, myristic, palmitic, oleic, linoleic, and linolinic. In'the case of the last three which are unsaturated, fatty acids hydrogenation cannot be used since this would reduce the olefinic unsaturation. Instead the peptide resin is treated with hydrogen fluoride under anhydrous conditions to remove all protecting groups and the corresponding acylated derivative of tuftsin hydrofiuoride. The yield with the unsaturated fatty acids for the final product of lipoyl-tuftsin is at the lower ranges, 48%55%. I

Purification of the final lipoyl products varied with the length of the chain. The main contaminant is uncoupled tuftsin. With acetyl-, propionyl and butyryl-tuftsin, the separation of the two is done on aminex column (Dowex 50) with a linear gradient of pyridine acetate buffer pH 4.0 to 6.0 with pyridine molarity gradient of 1.2 N-2.5 N. The first large peak to appear is the lipoyl tuftsin, the smaller free tu-ftsin appears later.

With higher chain fatty acids Dowex 10 chromatography with 0.1 M acetic acid suffices to separate the two. Tuftsin with a free NH group is strongly basic, binds to sephadex and is retarded, appearing as a small molecular weight substance with free amino acids. The isolated pyridinium salt is then converted to the sodium salt by base exchange.

EXAMPLE XII Formation of O(Thr)-stearoyl-tuftsin IV 0.5 mmole of N t-BOC-threonyl-N-CBZ-lysyl-proyly- N -nitroarginine on 1.5 g. resin is washed thoroughly with chloroform and methylene chloride and 1.5 mmoles of stearic acid in 1:1 DMF:chloroform is added. This is followed by 1.5 mmoles of carbonyl diimidazole in the same solvents. The reactants are rocked at room temperature in the same vessel used for the Merrifield synthesis of the peptide for two hours. The peptide is then cleaved with hydrogen bromide in trifluoroacetic acid for 1.5 hours at room temperature. Catalytic hydrogenation is then carried out and the resultant product purified as in the previous examples.

Other alkanoyl derivatives corresponding to the N- acylated derivative of the previous example are similarly prepared.

EXAMPLE XIII Formation of N-penicillinyl tuftsin IV EXAMPLE XIV Formation of O(Thr)-penicillinyl tuftsin IV This product is prepared utilizin the procedure of Example XII by reaction of 0.46 mmole of the starting protected peptide resin with 2.5 mmoles of penicillin using carbonyl diimidazole as the coupling agent in 1:1 chloroform:dimethylformamide.

EXAMPLE XV Preparation of Thr-Lys-Pro-Arg-Pro-Thr-Lys-Pro-Arg This nonapeptide is prepared utilizing the procedures of Example V. After the formation of Pro-Arg resin the coupling of each subsequent amino acid is efi'ected using three equivalents of the protected acid with respect to the starting arginine. After the completion'of each coupling reaction the product is washed with ethanol, chloro- 16 form and methylene. chloride. Deprotection is effected at each step with trifiuoroacetic acidand neutralization is with triethylamine. The nonapeptide is cleaved from the resin with hydrogen bromide intrifluoroaceticacid and the nitro arginine residues reduced by catalyst hydrogenation with palladium or barium sulfate.',,Pu rification is effected on Sephadex vG10 followed by an aminex column. i j

EXAMPLE XVI Preparation of tablets 1000 g. of the tuftsin IV and 2000 g. of lactose were thoroughly mixed together and the whole was passed through a 30 mesh sieve. II

A paste was separately prepared with 80 g. of corn starch and 350 ml. of distilled water.

The above mixture waswell kneaded with the paste and the mass was passed through a 4 mesh sieve and the resulting globules were dried at 50 C. for 15 hours.

The dried globules were then granulated first on a granulating machine and then passed through a 16 mesh sieve. The grains were covered with a powdery mixture which had been prepared by blending 30 g. of calcium stearate, 200 g. of cornstarch and g. of talc, and then passed through a 40 mesh sieve.

Tablets each containing 250 mg. of tuftsin IV were made of the above-obtained granules in accordance with the conventional procedure knownin the art.

EXAMPLE XV HI Preparation of an aqueous solution for oral administration A mixture consisting of:

Tuftsin IV hydrochloride g 20.0 Cane sugar g 100.0 Glycerine ml 100.0 Ethyl p-oxybenzoate g 1.5 Artificial orange essence 'ml 0.2 Essential oil of orange ml;. '1.0

was added to distilled water to make final volume. I I

What is claimed is: I I

1. Therapeutically useful non-antigenic polypeptides containing at least fourand up to about nineteen amino acids comprising one unit or repeating units each unit containing four to five amino acids at .least one of the terminal amino acids in each unit being a hydroxyl substituted amino acid, each repeating unit being joined to an adjacent unit by proline or'cystine, each unit containing two basic amino acids linked together by one to two proline molecules and pharmacologically acceptablesalts and derivatives thereof. 1

2. A polypeptide as in claim 1 containing 4 amino acids joined together to form Thr-Lys-Pro-Arg, Ser-Lys- Pro-Arg, Thr-LysPro-Lys, Thr-Arg-Pro-Arg, Thr-Arg- Pro-Lys, Thr-Arg-Pro-Orn, Thr-Lys -Pro-Orn, Thr-Lys- Pro-His, Ser-Lys-Pro-His, Arg-Pro-Lys-Ser, Lys-Pro-Lys- Thr, Arg-Pro-Arg-Thr, Lys-Pro-Arg Thr, Orn-ProArgup, 1000 ml. oftlie 'Th'r, .Orn-Pro -LysThr, Tis-Pro-Lys-Thr or His-Pro-Lys- Ser and pharmacologically acceptable salts and deriva- -tives thereof.

1 7 or Ser-Lys-Pro-Pro-Arg and pharmacologically acceptable salts and derivatives thereof.

4. A polypeptide as in claim 1 containing nine amino acids wherein the repeating unit is Thr-Lys-Pro-Arg and pharmacologically acceptable salts and derivatives thereof.

5. A polypeptide as in claim 4 wherein all of the amino acids are in the L-form and pharmacologically acceptable salts and derivatives thereof.

6. A polypeptide as in claim 1 containing nine amino acids wherein the repeating unit is Arg-Pro-Lys-Thr and pharmacologically acceptable salts and derivatives thereof.

7. A polypetide as in claim 6 wherein all of the amino acids are in the D-form and pharmacologically acceptable salts and derivatives thereof.

8. A polypeptide as in claim 1 containing four amino acids joined to form Thr-Lys-Pro-Arg and pharmacologically acceptable salts and derivatives thereof.

9. A polypetide as in claim 8 wherein all of the amino acids are in the L-form and pharmacologically acceptable salts and derivatives thereof.

10. A polypeptide as in claim 1 containing four amino acids joined to form Arg-Pro-Lys-Thr and pharmacologically acceptable salts and derivatives thereof.

11. A polypeptide as in claim 10 wherein all of the amino acids are in the D-form and pharmacologically acceptable salts and derivatives thereof.

12. Thr-Lys-Pro-Arg amide, lower esters and pharmacologically acceptable acid addition salts thereof.

13. Thr-Lys-Pro-Arg in which the hydroxyl group of the threonine moiety is esterified with an alkanoyl or alkenoyl group containing up to eighteen carbon atoms and pharmacologically acceptable salts and derivatives thereof.

14. Thr-Lys-ProArg in which the amino group of the threonine moiety is acylated with an alkanoyl or alkenoyl group containing up to eighteen carbon atoms and pharmacologically acceptable salts and derivatives thereof.

References Cited Ishii, Nip. Kag, Zass., 81 1586 (1960). Chem. Abstr. 56:5054c.

Krampitz et al.: Naturwiss. 54, 516 (1967). Najjar et 21.: Nature, 228, 672 (1970).

LEWIS GOTTS, Primary Examiner R. J. SUYAT, Assistant Examiner US. Cl. X.R. 

